The supply of viable tissues and cells for autologous implantation and heterologous transplantation (hereinafter jointly referred to as transplantation) and study is limited in part by the time a tissue or organ can be maintained in a viable state. Increasing the length of time that a tissue or organ remains viable may drastically increase the likelihood that a particular tissue or organ reaches a recipient or researcher in a viable state.
The transplantation of tissues, natural or engineered, including vascularized tissues and avascular tissues, including, but not limited to, vascular tissue, such as blood vessels, musculoskeletal tissue, such as cartilage, menisci, muscles, ligaments and tendons, skin, cardiovascular tissue, such as heart valves and myocardium, neuronal tissue, periodontal tissue, glandular tissue, organ tissue, islets of Langerhans, cornea, ureter, urethra, breast tissue, and organs, intact or sections thereof, such as pancreas, bladder, kidney, liver, intestine and heart, may all be benefited by increasing the length of time that such tissues and organs remain viable. In the present era of arterial replacement, at least 345,000-485,000 autologous coronary grafts (either arteries or veins) and over 200,000 autogenous vein grafts into peripheral arteries are performed each year. Report of a working party of the British Cardiac Society: Coronary Angioplasty in the United Kingdom. Br Heart J. 66:325-331, 1991; Heart and Stroke Facts: Statistical Supplement, American Heart Association, 1996; and Callow AD. xe2x80x9cHistorical overview of experimental and clinical development of vascular grafts,xe2x80x9d In: Biologic and Synthetic Vascular Prosthesis, Stanley J (Ed), Grune and Stratton, New York, 11, 1983. A recent marketing report indicated that at least 300,000 coronary artery bypass procedures are performed annually in the United States involving in excess of 1 million vascular grafts. World Cell Therapy Markets, Frost and Sullivan, 5413-43 Revision #1, ISBN 0-7889-0693-3, 1997.
Many of these patients do not have autologous veins suitable for grafts due to pre-existing vascular disease, vein stripping or use in prior vascular procedures. It has been estimated that as many as 30% of the patients who require arterial bypass procedures will have saphenous veins unsuitable for use in vascular reconstruction. Edwards W S, Holdefer W F, Motashemi, M, xe2x80x9cThe importance of proper caliber of lumen in femoral popliteal artery reconstruction,xe2x80x9d Surg Gynecol Obstet. 122:37, 1966. More recently it has been demonstrated that 2-5% of saphenous veins considered for bypass procedures were unusable on the basis of gross pathology and that up to 12% were subsequently classified as diseased. These xe2x80x9cdiseasedxe2x80x9d veins had patency rates less than half that of non-diseased veins. Panetta T F, Marin M L, Veith F J, et al., xe2x80x9cUnsuspected pre-existing saphenous vein disease: an unrecognized cause of vein bypass failure,xe2x80x9d J Vasc Surg. 15:102-112, 1992. However, we estimate that if all arterial grafts and alternative veins are utilized according to current surgical practice, the maximum number of potential allograft recipients is probably closer to 10%.
Vitrified arterial grafts may also have a market as a scaffold for the seeding and adhesion of autologous endothelial cells or genetically modified endothelial cells. Prosthetic grafts are currently employed for large diameter (greater than 6 mm internal diameter) non-coronary applications. Between 1985 and 1990, approximately 1,200 allogeneic vein segments were employed for arterial bypass. Brockbank K G M, McNally R T, Walsh K A, xe2x80x9cCryopreserved vein transplantation,xe2x80x9d J Cardiac Surg. 7:170-176, 1992. The demand for allogeneic veins is growing despite the well documented immune response to these grafts and the low clinical patency rates. In 1991 alone, at least 1,400 allograft saphenous vein segments were transplanted. McNally R T, Walsh K, Richardson W, xe2x80x9cEarly clinical evaluation of cryopreserved allograft vein,xe2x80x9d Proceedings of the 29th meeting of the Society for Cryobiology, Cryobio., Abstract #4, 1992. Conservatively, the market potential for vitrified vascular grafts may be 50,000 units per year, or 10% of all vascular grafting procedures in the United States.
Blood vessels are also a ubiquitous component of vascularized tissues and organs, both human and animal, which may one day be successfully stored by vitrification for transplantation. Providing that significant immunological issues can be overcome, animal-derived grafts may, one day, provide an unlimited supply of blood vessels and vascularized tissues and organs that could be stored in a vitrified state prior to transplantation.
Avascular tissues may also be used for transplantation. For example, on average, an orthopedic surgeon specializing in knee surgery will treat between 10-20 patients per year who have sustained traumatic, full-thickness articular cartilage injuries. These patients may all be candidates for cartilage implantation. Approximately 30% of all Anterior Cruciate Ligament (ACL) tears have an associated full-thickness cartilage defect that often is undetected, even after surgery. For example, it was estimated that 20.4% of the 392,568 patients who received cartilage repairs in 1996 were candidates for a cartilage implant.
Over time, most full-thickness defects deteriorate and cause significant joint impairment. Since cartilage is avascular, the recruitment of cells to aid healing of partial thickness defects is difficult. In contrast, full-thickness defects have the potential for partial healing when techniques such as abrasion arthroplasty are employed. Unfortunately, however, these procedures generally result in mechanically inferior fibrous scars.
Fresh osteochondral allografts have proven to be effective and functional for transplantation. The limited availability of fresh allograft tissues, however, necessitates the use of osteoarticular allograft banking for long-term storage. Although cryopreservation involving freezing is a preferred method for storing tissue until needed, conventional protocols result in death of 80-100% of the chondrocytes and damage to the extracellular matrix due to ice formation. These detrimental effects are the main obstacles preventing successful clinical outcome. Various studies using animal articular cartilage models and human cartilage biopsies have revealed no more than 20% chondrocyte viability following conventional cryopreservation procedures employing either dimethyl sulfoxide (DMSO) or glycerol as cryoprotectants. Such results greatly limit the possibilities for transplantation or grafting harvested cartilage.
Low temperature preservation of biological tissues and organs, i.e., cryopreservation, has been the subject of much research effort. Cryopreservation can be approached by freezing or by vitrification. If the organ or tissue is frozen, ice crystals may form within the organ or tissue that may mechanically disrupt its structure and thus damage its ability to function correctly when it is transplanted into a recipient. Organized tissues and organs are particularly susceptible to mechanical damage from ice crystals formed during freezing.
Even when all cryopreservation variables are controlled, there is a limit, which is largely a function of tissue volume and geometry (including any associated fluids and packaging), beyond which traditional cryopreservation methods do not consistently work. For example, in cryopreserved allograft heart valves, the leaflet fibroblasts survive well (70-90%), but neither the endothelial cells nor the smooth muscle cells of the aortic tissue associated with the valve survive. Cryopreservation can also be effective for isolated islets of Langerhans, but preservation of islets in bioengineered capsules can be technically difficult. Skin is relatively easy to preserve because of the thin, flat structure of the tissue. It appears that thawing of skin-like products, however, can be technically difficult due to the narrow window for error during warming, outside of which ice growth resulting in tissue damage can occur. In all of these examples, the problems are due to ice formation either within the cells, the extracellular matrix, the capsule, or, as in the case of heart valve endothelium, compression in the lumen of the associated artery.
Vitrification, by contrast, means solidification, as in a glass, without ice crystal formation. The principles of vitrification are well-known. Generally, the lowest temperature a solution can possibly supercool to without freezing is the homogeneous nucleation temperature Th, at which temperature ice crystals nucleate and grow, and a crystalline solid is formed from the solution. Vitrification solutions have a glass transition temperature Tg, at which temperature the solute vitrifies, or becomes a non-crystalline solid. Owing to the kinetics of nucleation and crystal growth, it is effectively impossible for water molecules to align for crystal formation at temperatures much below Tg. In addition, on cooling most dilute aqueous solutions to the glass transition temperature, Th is encountered before Tg, and ice nucleation occurs, which makes it impossible to vitrify the solution. In order to make such solutions useful in the preservation of biological materials by vitrification, it is therefore necessary to change the properties of the solution so that vitrification occurs instead of ice crystal nucleation and growth. It is also important that all viability and tissue function be maintained by the entire vitrification process.
While it is generally known that high hydrostatic pressures raise Tg and lower Th, vitrification of most dilute solutions by the application of pressure is often impossible or impractical. In particular, for many solutions vitrifiable by the application of pressure, the required pressures cause unacceptably severe injury to unprotected biomaterials during vitrification thereof. While it is also known that many solutes, such as commonly employed cryoprotectants like DMSO, raise Tg and lower Th, solution concentrations of DMSO or similar solutes high enough to permit vitrification typically approach the eutectic concentration and are generally toxic to biological materials.
One type of damage caused by cryoprotectants is osmotic damage. Cryobiologists learned of the osmotic effects of cryoprotectants in the 1950""s and of the necessity of controlling these effects so as to prevent damage during the addition and removal of cryoprotectants to isolated cells and tissues. Similar lessons were learned when cryobiologists moved on to studies of whole organ perfusion with cryoprotectants. Attention to the principles of osmosis were essential to induce tolerance to cryoprotectant addition to organs.
Despite efforts to control the deleterious osmotic effects of cryoprotectants, limits of tolerance to cryoprotectants are still observed. There appear to be genuine, inherent toxic effects of cryoprotectants that are independent of the transient osmotic effects of these chemical agents.
Studies by the present inventors and others have examined methods of controlling non-osmotic, inherent toxicity of cryoprotectant agents. The results indicate that several techniques can be effective alone and in combination. These include (a) the use of specific combinations of cryoprotectant whose effects cancel out each other""s toxicities; (b) exposure to cryoprotectants in vehicle solutions that are optimized for those particular cryoprotectants; (c) the use of non-penetrating agents that can substitute for a portion of the penetrating agent otherwise needed, thus sparing the cellular interior from exposure to additional intracellular agents; and (d) minimizing the time spent within the concentration range of rapid time-dependent toxicity.
Some of these techniques are in potential conflict with need to control osmotic forces. For example, reduced temperatures also reduce the influx and efflux rate of cryoprotectants, thereby prolonging and intensifying their osmotic effects. Similarly, minimizing exposure time to cryoprotectants maximizes their potential osmotic effects. Thus, there must be a balance reached between the control of osmotic damage and the control of toxicity. Means for obtaining this balance are described in U.S. Pat. No. 5,723,282 to Fahy et al. However, this patent does not describe a particular method to be used for blood vessels, cartilage or other tissues for which vitrification offers a potential technique for improved crypreservation. In addition, this patent does not discuss any protocols for cooling or warming the organ or tissue.
The present invention is directed to a method for vitrification of tissues, including vascularized tissues and avascular tissues, or organs. The method comprises immersing the tissue or organ in increasing concentrations of cryoprotectant solution at a temperature greater than xe2x88x925xc2x0 C. to a cryoprotectant concentration sufficient for vitrification; rapidly cooling the tissue or organ to a temperature between xe2x88x9280xc2x0 C. and the glass transition temperature (Tg); and further cooling the tissue or organ from a temperature above the glass transition temperature to a temperature below the glass transition temperature to vitrify the tissue or organ.
The present invention is also directed to a method for removing a tissue or organ from vitrification in a cryoprotectant solution. The method comprises slowly warming a vitrified tissue or organ in the cryoprotectant solution to a temperature between xe2x88x9280xc2x0 C. and the glass transition temperature; rapidly warming the tissue or organ in the cryoprotectant solution to a temperature above xe2x88x9275xc2x0 C.; and reducing the concentration of the cryoprotectant by immersing the tissue or organ in decreasing concentrations of cryoprotectant.
The present invention is also directed to a method for treating tissues or organs such that viability is retained at a high level. For example, for blood vessels, the invention provides that smooth muscle functions and graft patency rate are maintained. In particular, the present invention is directed to a method in which at least 50%, preferably at least 70%, and more preferably greater than 80% of smooth muscle functions and graft patency rate are maintained relative to fresh untreated controls.